Experimental determination of the phosphorylation state of phenylalanine hydroxylase.
A monoclonal antibody (PH 7), which recognizes the phosphorylated form of phenylalanine hydroxylase from human liver, has been used for the analysis of the enzyme in crude cell extracts from rat. In immunoblot analyses of rat liver cell extracts, the extent of binding of PH 7 closely correlates with the phosphorylation state of phenylalanine hydroxylase, as judged by [32P]Pi incorporation. These observations have made possible the rapid non-radioactive quantification of hormonal effects on phenylalanine hydroxylase phosphorylation state. In particular, the glucagon-dependent phosphorylation of phenylalanine hydroxylase in liver cells was investigated. Epidermal growth factor was shown to modulate this process. In addition, this technique was used to demonstrate, for the first time, that dibutyryl cyclic AMP, unlike the Ca2+ ionophore A23187, stimulates the phosphorylation of phenylalanine hydroxylase in isolated kidney tubules from rat.
Green
A K
AK
Department of Biochemistry, University of Liverpool, U.K.
Cotton
R G
RG
Jennings
I
I
Fisher
M J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005934:Glucagon / Q000494:pharmacology; D007684:Kidney Tubules / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D004815:Epidermal Growth Factor; D005934:Glucagon; D010651:Phenylalanine Hydroxylase